The present invention generally relates to an arrangement and method for performing chromatography. The present invention particularly relates to an arrangement and method for performing chromatography utilizing electroosmotic flow of a mobile phase.
Multiple techniques have been developed which enable the separation of complex mixtures into their components. Chromatography is one such technique. Chromatography can be described as a separation process based on differences in the rate at which the components of a mixture move through a chromatographic bed under the influence of a mobile phase which moves relative to a chromatographic bed. The chromatographic bed will typically include a plurality of porous or microporous particles, such as bonded C18 silica, wherein the collective surface of these particles make up the stationary phase. Several types of chromatography systems have chromatographic bed packed into the interior of a column. Alternatively, the chromatographic bed can be dispersed on a glass plate. An example of a chromatography system that utilizes the chromatographic bed packed into a column is High Performance Liquid Chromatography (hereinafter referred to as HPLC). An example of a chromatography system that utilizes the chromatographic bed dispersed on a glass plate is Thin Layer Chromatography (hereinafter referred to as TLC) or Overpressurized Layer Chromatography (hereinafter referred to as OPLC).
As previously mentioned HPLC involves packing the chromatographic bed within the interior of a column. The mobile phase is then pumped through the column (and thus through the chromatographic bed) at a very high pressure. A sample is then introduced into the chromatographic system and is pumped through the chromatgraphic bed. As the sample is pumped through the chromatographic bed the components of the sample are partitioned between the mobile phase and the stationary phase based upon their differing physical and chemical characteristics. For example, the components of the mixture can be partitioned between the mobile and stationary phases based upon their polarity, charge, and size. Since the components of a mixture will typically differ based upon their polarity, charge, and size they can be separated from each other by advancing them through the chromatographic bed.
HPLC is a very useful chromatographic technique, however it does suffer from several disadvantages. For example, (i) HPLC system can only separate one mixture at a time, (ii) HPLC systems require special pumps and inlet devices to respectively generate and accommodate the high pressures required to perform HPLC, (iii) HPLC columns must be constructed from mechanically strong materials which limits the use of glass columns that are particularly useful for handling many biological samples, (iv) HPLC systems designed for preparative chromatography techniques are very expensive, and (v) detector dead volumes must be keep extremely small (several microlitres) in order to avoid additional band spreading.
With respect to TLC, the chromatographic bed is a layer (0.1-0.5 mm thick) of a sorbent material spread uniformly over the surface of a glass or plastic plate. The mixture to be separated is applied to the chromatographic bed with a micropipette and dried. The TLC plate is then placed in a chamber so that a small portion of the stationary phase is in contact with a mobile phase. The TLC plate is developed by allowing the mobile phase to ascend up the plate by capillary action. The basis for the separation of the mixture into its respective components is the same as discussed above with respect to HPLC, i.e. the components are separated due to their different partitioning between the stationary and mobile phases. This in turn is based upon the differing polarity, charge, and size characteristics of each of the components of the mixture to be separated.
However, like HPLC, TLC also suffers from several significant disadvantages. In particular, the separation efficiency by TLC is limited by the inadequate mobile phase flow under capillary action. This capillary-induced mobile phase flow is neither fast enough nor constant throughout the chromatographic run, and both of these drawbacks tend to decrease the separation efficiency of TLC substantially. Moreover, the relatively slow movement of the mobile phase results in rather long development times.
OPLC attempts to overcome the aforementioned difficulties associated with TLC. This technique forces the mobile phase through the chromatographic bed disposed on the plate by applying high pressure to the mobile phase. This results in a flow rate that can be controlled and remains constant throughout the development of the plate. A consequence of the constant flow rate is that the number of theoretical plates encountered by a solute will increase linearly with increasing migration distance. In addition, the total time of an analysis is substantially decreased because the mobile phase flows faster.
OPLC also suffers from significant drawbacks. In particular, the flow of the mobile phase in OPLC systems is laminar. Laminar flow profile or parabolic flow profile means that throughout the cross-sectional area of the mobile phase within a channel between particles the center portion of the liquid of the mobile phase flows faster than the liquid close to the wall of the channel. The laminar flow profile of OPLC systems results in migration characteristics of the mobile phase being sensitive to the particle size and the particle size distribution of the stationary phase. Having the migration characteristics of the mobile phase sensitive to the particle size and the particle size distribution of the stationary phase can decrease the separation efficiency of OPLC.
What is needed therefore is a chromatographic arrangement and method which overcomes one or more of the aforementioned problems.